1
Q
Characteristics of signalling pathways
A
‣ have multi-layered, hierarchical structure.
‣ amplify initial signal.
‣ often regulate multiple cellular functions in response to single signal.
‣ tightly controlled due to constitutive and regulated feedback mechanisms.
‣ often desensitization during continued presence of signal.
2
Q
Intracellular signalling is initiated by what?
A
A ligand that binds to a receptor protein.
3
Q
What does the last intracellular signalling proteins do?
A
They altes the structure and function of a target protein leading to a cellular response to the initial stimulus.
4
Q
Example of regulating multiple cellular functions in response to single signal
A
The signalling pathway elicits changes in the activity of a transporter leading to changes in ion transport.
But it also alters the activity of a metabolic enzyme leading to a change in the metabolism of the cell.
At the same time, the signalling pathway affects the activity of a gene regulatory protein, leading to changes in gene expression.
Pathway activation also alters the propensity of a cytoskeletal protein to bind to other cytoskeletal proteins, leading to changes in cell shape.
Cell cycle proteins cause altered cell growth and division.
5
Q
Signalling divergence
A
The ability of signalling pathways to affect multiple cellular changes.
6
Q
How do signalling pathways amplify initial signal?
A
A ligand binding to its cognate receptor is often able to activate multiple copies of this initial intracellular signalling protein, and one of the signalling proteins is then able to activate multiple copies of more signalling proteins. An individual ligand molecule is thus able to cause a very sustained and robust change in cellular function.
7
Q
Desensitization
A
Signalling pathways are frequently shut down even before a ligand dissociates from its receptor. Even in the continuous presence of a ligand, the change in cell function is often not sustained. This phenomenon can be due to feedback mechanisms in which downstream components of the signalling pathway inhibit upstream components. Alternatively, individual components of the signalling pathway, such as the receptor for the ligand, may no longer be active because they undergo some kind of conformational change that renders them inactive.
8
Q
The types of signalling
A
Synaptic
Contact-mediated
paracrine
autocrine
endocrine
9
Q
Synaptic signalling
A
Neurotransmitters are released at synapses via a regulated exocytosis. At this synaptic junction, the target cell expresses receptors on its plasma membrane. Neurotransmitter binding to these receptors initiates an intracellular signalling pathway.
10
Q
cell junctions between neurons called
A
synapses
11
Q
Neurons secrete ligands called
A
neurotransmitters
12
Q
Contact-mediated
A
Ligands activating receptors on the target cells are membrane-bound proteins. These membrane bound proteins of the signalling cell can then bind to receptors on the surface of the target cell and that initiates intracellular signalling in the target cell.
13
Q
Paracrine
A
Ligands are secreted not at specialized cell junctions, but they are secreted into the extracellular milieu. The ligand now needs to diffuse over short distances to neighbouring cells to bind on receptors expressed on the surface of these cells. An individual cell emitting a paracrine signal can often activate multiple target cells.
14
Q
The receptors on target cells in paracrine signalling often have what?
A
High affinity for the ligand, allowing them to detect low concentration of this ligand.
15
Q
Paracrine signalling the ligand is often called what?
A
A local hormone
16
Q
Autocrine
A
The signalling cell is also the target cell. The ligand released by the signalling cell can act on receptors expressed on its surface, initiating a signalling pathway inside in the cytoplasm.
17
Q
What does autocrine signalling do most of the time?
A
Happens in conjunction with synaptic signalling or paracrine signalling. While the ligand exocytosed by the signalling cell activates one or more nearby target cells, it also binds to receptors on the signalling cell. This binding leads to a negative feedback that prevents further release of the ligand by the signalling cell.
18
Q
Endocrine
A
A hormone is secreted by an endocrine cell. The hormone enters the bloodstream and is disseminated throughout the body. Hormones leave the bloodstream and diffuse through tissues until they encounter a target cell expressing receptors for this hormone. The target cells are often far away from the endocrine cell. They can detect the hormone released as long as they express receptors with a very high affinity for the hormone to allow detection of low concentrations of this hormone in the periphery.
19
Q
The ligand is called what in endocrine signalling
A
Hormone
20
Q
Signalling cell is called what in endocrine signalling
A
An endocrine cell
21
Q
Types of receptors
A
Ionotropic receptors
Metabotropic receptors
Enzyme-linked receptors
Intracellular and nuclear receptors
22
Q
Ionotropic receptors
A
Are ligand-gated ion channels and typically consist of multiple polypeptides that each span the plasma membranes several times. These polypeptides, aka receptor subunits, are grouped around a central ion pore with multiple subunits.
23
Q
How do ionotropic receptors work?
A
This ion pore is normally closed and only the binding of a ligand to the ligand binding domain of the receptor opens the channel pore, allowing ions to diffuse across the plasma membrane, according to their electrochemical gradient. This creates a membrane potential change in the target cell.
24
Q
Ionotropic receptors are either what?
A
cation channels or anion channels
25
Cation channels
often conduct sodium and potassium ions or sodium, potassium and calcium ions
26
Anion channels
are overwhelmingly chloride channels
27
Many neurotransmitter receptors are what?
Ionotropic and metabotropic
28
Metabotropic receptors are also called what?
G protein-coupled receptors (GPCRs)
29
Metabotropic receptors
They activate intracellular proteins that are called G proteins through ligand binding. In their inactive state metabotrophic receptors are bound to inactive G proteins. Once a ligand binds to the ligand binding domain of the metabotropic receptor, the receptor can activate the G protein that is bound to it. The G protein then can activate downstream effector proteins, initiating a signal transduction cascade.
30
What do metabotropic receptors have?
Consist of a single polypeptide that spans the membrane seven times (aka 7 transmembrane domains)
31
Enzyme-linked receptors
Are transmembrane proteins that have an extracellular ligand-binding domain and an intracellular enzymatically active domain.
Under basal conditions, this enzymatic domain is inactive. When ligand binds to the ligand-binding domain the conformation of the receptor changes and the enzymatic domain becomes active.
They can also have other enzymatic activities. For instance, they can generate cyclic GMP, or act as proteases that are able to cleave specific peptide bonds in their target proteins, among other enzymatic activities.
32
The enzymatic activity of enzyme-linked receptors varies between what?
Subclasses.
33
A large subclass of enzyme-linked receptors are?
the receptor tyrosine kinases.
34
Kinases
enzymes that can phosphorylate amino acid residues and target proteins that have hydroxyl groups.
35
tyrosine kinases
Tyrosine kinases phosphorylate hydroxyl groups of tyrosine residues in their target proteins, causing conformational alteration that changes its enzymatic activity or alters its ability to bind to other proteins.
36
Enzyme-linked receptors subclasses:
- receptor tyrosine kinases
- serine kinases
- tyrosine and S/T phosphatases
- guanylyl cyclases etc
37
Serine Kinases
They phosphorylate serine or threonine residues in their target proteins, changing their structure, leading to altered enzymatic activity or binding affinity for other proteins.
38
S/T Phosphatases
They catalyze the inverse reaction: They dephosphorylate target proteins - either at tyrosine residues, if they are tyrosine phosphatases, or at serine and threonine residues, if they are serine phosphatases. By dephosphorylating their target proteins they restore them to their original state.
39
A very important member receptor tyrosine kinase subclass is what?
a receptor that binds neurotrophic factor or NGF
40
NGF
A local peptide hormone that is released into the extracellular milieu by neurons. NGF molecules are dimers that can bind not only one but two receptors.
41
NGF receptor binding
If it binds, NGF brings two receptors into close contact. The enzymatic domains of the two NGF receptors are now in close proximity, allowing the kinase domain of one receptor to phosphorylate the intracellular domain of the other NGF receptor and vice versa. This allows effector proteins to bind to the receptor. These effector proteins only bind when the receptor is phosphorylated. The effector proteins normally reside in the cytosol and are functionally inactive. Binding to the NGF receptor, however, activates them, allowing them to initiate signal transduction pathways (via G-proteins)
42
Dimerization
the process where two individual molecules (monomers) combine to form a single dimer, which is a larger complex made of two subunits.
43
Intracellular receptors and nuclear receptors are where?
These receptors do not reside in the plasma membrane, they are in the cytosol or nucleus. So, they need to be activated by ligands that are very lipophilic and can diffuse across the plasma membrane without any help from other proteins.
44
Intracellular receptors and nuclear receptors are what?
Are transcriptional modulators: they activate or repress the transciption of a set of target genes. Ligand binding allows them to bind to certain DNA sequences and alter mRNA transcription.
Examples: the glucocorticoid receptor and the thyroid hormone receptor.
45
G proteins and their common features
Intracellular signalling proteins. They all can bind GTP and hydrolyze it. They can cleave the terminal phosphodiester bond and generate GDP and phosphate. In their GTP bound form, G Proteins can bind effector proteins and activate them. Hydrolysis of their GTP leads to G protein inactivation. The G protein dissociates from the effector protein. G proteins can be recycled by exchanging the GDP in the inactive G protein for GTP. This activates the G protein and initiates another round of signal transduction.
46
Two classes of G proteins
Heterotrimeric G proteins and Small monomeric G proteins
47
Heterotrimeric G proteins
Composed of three distinct subunits or polypeptides (α, β, γ); often activated by metabotropic receptors.
48
Small monomeric G proteins:
Single polypeptide; activation by receptor tyrosine kinases and other mechanisms (afferent signalling proteins).
49
Heterotrimeric G proteins activation
They bind in their inactivated GDP-bound state to metabotropic receptors. Ligand binding to the GPCR causes a conformational change in the receptor that is transduced to the G protein. That allows GDP to dissociate from the G protein, and the binding of GTP. GTP binding activates the G protein whose subunits now dissociate from the metabotropic receptor. The alpha subunit and the beta/gamma subunits can diffuse independent of each other along the plasma membrane.
50
Alpha Subunits bind and activate what?
enzymes that can synthesize second messengers
51
beta/gamma bind and activate to what?
Ion channels, causing them to open.
52
The alpha subunit and the beta/gamma subunits are both associated to the membrane by what?
lipid anchors
53
The binding of G proteins modulates
the likelihood with which these ion channels open their channel pore.
54
Heterotrimeric G proteins inactivation
Bound to the effector, the alpha subunit of the heterotrimeric G protein can interact with another protein called the GTPase activating protein (GAP). GAP stimulates the GTPase domain in the alpha subunit, which then hydrolyzes the bound GTP. This inactivates the alpha subunit, which dissociates from its effector and re-associates with the beta/gamma subunits. The inactive heterotrimeric G protein will then bind to a metabotropic receptor ready to be activated again once a ligand binds to the receptor.
55
Heterotrimeric G proteins are sub-categorized into four individual groups which show what?
considerable specificity with respect to the target proteins they modulate.
56
Metabotropic receptors also show strong specificity towards what?
different heterotrimeric G proteins. Thus, any given metabotropic receptor can usually only activate one kind of heterotrimeric G protein, activation of any given metabotropic receptor specifically induces one particular signalling pathway.
57
The 4 heterotrimeric G proteins
Gs, Gi, Gt (transducin), Gq.
58
Gs protein
Activates an enzyme called adenylyl cyclase. This enzyme generates cyclic adenosine monophosphate or cAMP, which is a second messenger. Thus, a consequence of the activation of a Gs protein is a cytoplasmic rise in cAMP.
59
Gi protein
Also interact with adenylate cyclase, but they inhibit this enzyme leading to a drop in cAMP levels.
60
Gt (transducin)
It activates the effector cGMP phosphodiesterase. This enzyme cleaves and inactivates the second cyclic guanosine monophosphate or cGMP, leading to a drop in its (cGMP) concentration upon activation of transducin.
61
Gq
They activate the enzyme phospholipase C, which generates two second messengers, diacylglycerol (DAG), and inositol trisphosphate (IP3).
The activation of Gq leads to rises in the concentration of these two second messengers.
62
Monomeric G proteins
The GEF facilitates the replacement of GDP for GTP in the monomeric G protein. The GTP-bound G protein can also interact with GAP, which facilitates the hydrolysis of GTP by the protein and inactivation.
The G protein is then in it GDP-bound, inactive form and needs a GEF to become activated again. When bound to the receptor tyrosine kinase, some adaptor proteins are able to bind and activate GEFs, which can then activate monomeric G proteins. Thus, ligand binding to the receptor tyrosine kinase leads to G protein activation.
63
The activity of GAPs (for monomeric g proteins) can also be regulated by what?
Upstream signalling, placing the inactivation of G proteins under tight control.
64
Monomeric G proteins need what for activation?
a guanine nucleotide exchange factor or GEF
65
GEF is activated by what?
A variety of signals (including receptor tyrosine kinases).
66
the GEF does for monomeric G proteins what ______ does for heterotrimeric G proteins
metabotropic receptor
67
When can monomeric G proteins be regulated?
This activity cycle of monomeric G proteins can be regulated both at the stage of activation by GEF proteins and at the stage of inactivation by GAPs.
68
Three families of monomeric G proteins
ras, rho, rab
69
Ras family
Function: the regulation of cell proliferation, cell differentiation and cell survival. They activate the MAP kinase pathway.
70
Ras in cancer
Somatic mutations that lead to constitutively active forms of ras can lead to aberrant cell proliferation and tumour formation. In the nervous system, they are often required early on in development, where they allow for the proper expansion, differentiation, and survival of neurons.
71
Rho Family
They affect actin dynamics and are important for regulating cell shape. In neurons, they are necessary to regulate the outgrowth of neurites, axons and dendrites, and are involved in synapse formation and plasticity.
Effectors of rho-family G proteins are the serine/threonine kinases: PAK and ROCK. PAK and ROCK have effectors that are involved in actin polymerization.
72
Rab family
Function: regulating membrane trafficking, including vesicle formation, vesicle movement along actin and tubulin networks, and membrane fusion. Have various effectors.
73
MAP kinase pathway start
Starts with the activation of a variety of receptor tyrosine kinases. Ligand binding to these kinases causes, receptor dimerization and receptor phosphorylation, which allows for the recruitment of adaptor proteins such as the protein GRB2. These adaptor proteins can in turn recruit and activate guanine nucleotide exchange factors, which are necessary to activate small monomeric G proteins of the ras family by exchange of GTP for GDP. Activated ras can activate the so-called MAP kinase pathway.
74
MAP kinase pathway ending
MAP kinase itself must be phosphorylated to be active and requires a specific kinases, MAP kinase kinase, to be activated. MAP kinase kinase is also a serine/threonine kinase that looks and behaves a lot like MAP kinase. It needs to be phosphorylated by yet a third kinase, MAP kinase kinase kinase, to be able to phosphorylate and activate MAP kinase. MAP kinase kinase kinase is activated by an activated ras G protein.
75
MAP kinase
A kinase that can phosphorylate specific serine and threonine residues in its target proteins to alter their conformation and activity.
76
MAP kinase kinase
serine/threonine kinase that looks and behaves a lot like MAP kinase
77
Why did nature invent this complicated pathway with three kinases that activate each other?
Signal amplification: Each tyrosine kinase receptor can only activate few ras proteins, and each ras protein only one MAP kinase kinase kinase. However, each MAP kinase kinase kinase can phosphorylate many MAP kinase kinase proteins, and each MAP kinase kinase can phosphorylate many MAP kinase proteins, thus greatly amplifying the initial signal.
78
Second messengers
small cytosolic or membrane-bound molecules that can readily diffuse within the cell and activate downstream effectors.
79
4 classes of second messengers
cAMP, cGMP, IP3 and DAG, calcium.
80
cAMP (cyclic adenosine monophosphate)
Adenylyl cyclase generates cAMP out of ATP. cAMP is a water-soluble second messenger that can diffuse in the cytoplasm and can bind to and activate a number of effector proteins (PKA). However, cAMP can also signal more directly by binding to other effector proteins, such as cyclic nucleotide-gated ion channels. Binding of cAMP to these ligand-gated ion channels modulates their ability to conduct ions. In many neurons and specialized sensory receptor cells, cyclic nucleotide-gated ion channels have an outsize influence in regulating the membrane potential of the cell.
81
cAMP structure
It is an adenine-containing nucleotide that has a cyclic phosphodiester bond between a sole phosphate group and the ribose of the adenosine.
82
protein kinase A
a serine/threonine kinase that has many target proteins, for example metabolic enzymes, ion channels and ion transporters, gene transcription factors and so on.
83
PKA signalling regulates what?
Important metabolic pathways, such as glycolysis and the lipid metabolism. In neurons, it is intimately involved in the regulation of synaptic transmission.
84
cAMP phosphodiesterase
cAMP is degraded by this enzyme. The phosphodiesterase cleaves the 3’ phosphodiester bond of cAMP, generating the inactive adenosine monophosphate or AMP. AMP can then be recycled to form cAMP again. cAMP phosphodiesterase activity is enhanced following phosphorylation by PKA. This is an example of a negative feedback loop that makes sure that the pathway in question is quickly turned off.
85
cGMP (cyclic guanosine monophosphate) activation
It is generated by an enzyme called guanylyl cyclase from GTP. Unlike adenylyl cyclase, guanylyl cyclase is not regulated in its activity by a G protein. Guanylyl cyclase is what is often called “constitutively active”, meaning that it makes cGMP all the time. cGMP can activate several effector proteins: It activates protein kinase G. It binds and modulates conductance of cyclic nucleotide-gated channels, which mediate cGMP’s effects on membrane potential.
86
cGMP structure
The only structural difference between cAMP and cGMP is the purine connected to the ribose: In the case of cAMP it is adenine, in cGMP it is guanine. Both cGMP and cAMP contain the cyclic phosphate group that is connected to the 3’ and 5’ hydroxyl groups of the ribose via phosphodiester groups.
87
protein kinase G
a serine/threonine kinase that is structurally related to protein kinase A and mediates many effects of cGMP signalling.
88
cGMP inactivation
GMP is inactivated by an enzyme called cGMP phosphodiesterase. In contrast to the cAMP phosphodiesterase, this enzyme is NOT constitutively active. Rather, it needs to be activated by the alpha subunit of a heteromeric G protein, transducin. Because of this regulation of degradation rather than synthesis for the second messenger cGMP, a signal causes a reduction, rather than an increase in the second messenger cGMP.
89
cGMP
an important second messenger in photoreceptors, where it regulates the membrane potential: In the dark, cGMP is plentiful, because guanylyl cyclase generates cGMP constitutively and the cGMP phosphodiesterase is inactive. cGMP binds to cyclic nucleotide-gated channels, allowing for sodium influx and depolarization of the plasma membrane.
When light enters the eye, a G protein-coupled receptor rhodopsin, activates the G protein transducin, which activates cGMP phosphodiesterase, reducing cGMP levels. Cyclic nucleotide-gated channels close, and the membrane hyperpolarizes.
90
Phospholipase C
enzyme that is activated by a heterotrimeric G protein Gq and generates two second messengers: DAG and IP3.
91
IP3
a water-soluble second messenger that can diffuse freely in the cytosol.
92
DAG
A lipid and stays within the plasma membrane. DAG activates proteins that are located in or close to the plasma membrane.
93
PIP2
Phosphatidylinositol bisphosphate is a phospholipid in the Plasma membrane. PIP2 is cleaved at the phosphodiester bond to liberate IP3 and DAG
94
Phospholipase C needs what
PIP2
95
Phospholipase C (PLC) isoforms
* Major isoform activated by heterotrimeric G proteins (Gq).
* Activation of other isoforms by tyrosine kinases, calcium.
96
What does IP3 do?
Binds to and activates/opens IP3 receptors, ligand-gated calcium channels in the ER membrane (it's like a ligand). This allows the diffusion of calcium ions from the ER lumen, where the calcium concentration is relatively high into the cytoplasm.
97
Calcium released from ER stores is what?
“third messenger”, initiates Ca2+-
dependent signalling
98
DAG does what
diffuse laterally within the plasma membrane and binds to proteins that are either in the plasma membrane associated with the plasma membrane. One of those proteins is a serine/threonine kinase called protein kinase C.
99
What is needed to activate Protein kinase C?
DAG and calcium
100
Protein kinase C
a Ser/Thr kinase phosphorylating enzymes, ion channels, structural protein.
101
Basal Ca2+ concentration in
cytosol is what?
Low. Less than 100 nM. This is due to a very effective extrusion of calcium into the extracellular space by the plasma membrane calcium ATPase or PMCA, sometimes aided by the calcium/sodium exchanger. Calcium is transported from the cytosol into the endoplasmic reticulum by the sarcoplasmic/endoplasmic reticulum calcium ATPase or SERCA. Both extrusion into the extracellular milieu and sequestration in the ER are very effective. The concentration of free, unbound calcium ions is kept low by so-called calcium binding proteins (this is by buffering).
102
calcium binding proteins
proteins in the cytoplasm whose main function it is actually to bind calcium in the cytosol. They don’t participate in signalling themselves; they are not effector proteins.
103
How can calcium be increased in the cytoplasm?
through ion channels. Many ionotropic neurotransmitters allow the diffusion of calcium into the cytoplasm and that would not only lead to a change in membrane potential, but would also allow for calcium-dependent signalling to occur. Also, voltage-gated ion channels can allow for the influx of calcium ions from the extracellular space (through plasma membrane). But the extracellular space is not the only source of calcium ions. Calcium ions can also enter the cytosol from the ER. IP3 and ryanodine receptors (ER)
104
Ryanodine receptors
the ligand opening it is calcuim. So it allows for calcium-induced calcium release. For example, the influx of calcium ion in response to opening of voltage-gated calcium channels is often amplified by release from the ER through ryanodine receptors that open as a consequence of the initial rise in calcium concentration. This boosts the initial calcium signal increasing the response of calcium-dependent effector proteins to the signal.
105
Calcium signalling
It can activate effector proteins by first binding to calcium sensors, small proteins that can, in their calcium-bound state, interact and regulate effector proteins.
106
Calmodulin
Calcium sensor. It can bind to and activate a large number of downstream effector proteins, like serine/threonine kinase called calcium/calmodulin-dependent kinase. It can activate phosphatases such as protein phosphatase 2B.
107
calcium/calmodulin-dependent kinase (CaMKII)
A kinase phosphorylates many effector proteins and is involved in the plasticity of synaptic transmission and gene regulation, among other functions.
108
protein phosphatase 2B
which dephosphorylate target proteins to return them to their basal conformational and activity state. It depends on a multitude of factors, such as the amplitude of the calcium signal (i.e. the calcium concentration), the duration of the signal, and its exact location within the cell whether more kinase or phosphatase get activated.
109
receptors, G proteins and second messenger systems has what characteristics?
usually effects transient changes, brought about by posttranslational modification of effector proteins, such as phosphorylation. They lead to short-lasting changes in cellular function, such as increased transport of an ion species across the plasma membrane or upregulation of a metabolic pathway to meet an immediate energy demand.
110
Regulation of gene expression by signal transduction pathways
Synthesis of new mRNA and protein regulated by signal transduction pathways. Slow onset (>30 min), long-lasting (days wheres something like phosphorylation is hours). Gene transcription requires binding of transcriptional activator proteins to DNA near promoter of target gene. inding of transcriptional activator allows formation of RNA polymerase complex, transcription of gene.
111
Why is mRNA making slow
because it involves several time-consuming steps: mRNA transcription itself, the export of mRNA out of the nucleus, the translation of mRNA to obtain a nascent protein, and the folding and cellular targeting of the protein. Therefore, any signalling that involves gene expression is slow.
112
Each transcriptional activator controls what?
the transcription of a small subset of inducible genes, usually around tens to hundreds, which are transcribed simultaneously when a particular transcriptional activator gets activated.
113
transcriptional repressors
proteins that bind DNA and prevent the recruitment of general transcription factors and RNA polymerase. sometimes signal transduction events can prevent transcription of a gene by activating a transcriptional repressor, or, alternatively, elicit transcription of a gene by inhibiting a transcriptional repressor. Moreover, sometimes different transcriptional activators and repressors interact to precisely control the expression of a certain target gene.
114
What is the primary function of the CREB protein, and what DNA element does it bind to?
Function: CREB is a transcriptional activator.
DNA Element: It binds to cAMP-Responsive Elements (CRE), which are regulatory DNA sequences near the promoters of genes it controls
115
How is CREB's activity as a transcriptional activator primarily regulated?
CREB must be phosphorylated to be active. Phosphorylation enables it to:
--Bind to its target CRE DNA sequence.
--Effectively recruit transcription factors and RNA polymerase.
116
Name the three key kinases that can phosphorylate and activate CREB.
Protein Kinase A (PKA)
Calcium-Calmodulin Dependent Kinase (CaMK)
MAP Kinase (via the Ras-MAPK pathway)
117
What cellular signals trigger CREB activation via its different kinases?
PKA: cAMP signaling.
CaMK: Transient elevations in calcium concentrations.
MAPK: Stimulation of Receptor Tyrosine Kinases
118
Why is CREB critical for neurons experiencing increased activity? Name two specific functional outcomes.
1. Increased Neurotransmitter Synthesis:
--Upregulates genes for neuropeptides.
--Increases expression of Tyrosine Hydroxylase, the rate-limiting enzyme for catecholamines (dopamine, norepinephrine, epinephrine).
2.Synaptic Plasticity:
--Supports learning and memory by regulating genes required for the formation and maintenance of synaptic connections.
119
Besides direct structural and metabolic genes, what other important transcriptional regulator does CREB activate?
CREB regulates the expression of c-Fos, another transcriptional activator. This creates a cascade that can amplify and diversify the transcriptional response.
120
What determines which signaling pathway (PKA, CaMK, or MAPK) is most effective at activating CREB-mediated transcription?
The effectiveness depends on:
--The specific neuronal cell type.
--The situation, including the location of signaling within the cell and the developmental state of the cell.
--Pathways often act synergistically for maximal activation.
121
What is the c-Fos protein, and what is its concentration in an unstimulated cell?
c-Fos is a transcriptional activator.
In unstimulated cells, it is present at very low concentrations, keeping its target genes inactive
122
How is the transcription of the c-Fos gene itself activated
The c-Fos gene has upstream activating sequences (UAS).
When CREB is activated (e.g., by PKA, CaMK, or MAPK), it binds to these UAS.
CREB then recruits general transcription factors and RNA polymerase, inducing c-Fos transcription and translation (new c-fos mRNA), leading to a strong increase in c-Fos protein.
123
How does the newly synthesized c-Fos protein activate its target genes?
c-Fos binds to a constitutively expressed transcriptional activator, c-Jun.
The c-Fos/c-Jun complex then binds to upstream activating sequences in a whole new set of inducible genes, upregulating their expression.
124
What is an "immediate early gene," and why does c-Fos fit this definition?
Definition: A gene that is one of the first to be transcribed in response to a cellular signal, without the need for new protein synthesis.
c-Fos: It is a primary target of signaling pathways (PKA, CaMK, MAPK) via CREB, making it one of the first genes activated, distinguishing it from the "late" genes it subsequently induces.
125
What are the two major functional advantages of using c-Fos as an intermediate?
Signal Amplification: A single activated CREB can lead to multiple c-Fos proteins, which can then activate a much larger number of target genes.
Signal Divergence: c-Fos allows the initial signal (e.g., from PKA) to regulate a broad set of genes different from those regulated directly by CREB
126
How is c-Fos expression used experimentally, and what is its broader biological role?
Experimentally: c-Fos expression is a marker to identify neurons that have undergone strong activation, typically involving large increases in cytoplasmic calcium and CaMK activation.
Biologically: Along with CREB, c-Fos is one of the most prominent transcriptional activators in neurons, crucial for converting transient signals into long-term changes in gene expression.
127
What are the three catecholamine neurotransmitters, and what is the name of the rate-limiting enzyme in their synthesis?
Catecholamines: Dopamine, Norepinephrine, and Epinephrine.
Rate-Limiting Enzyme: Tyrosine Hydroxylase (TH).
128
How is catecholamine synthesis quickly increased to meet demand during high neuronal activity?
Tyrosine Hydroxylase (TH) is phosphorylated.
Phosphorylated TH is more processive, generating more catecholamine precursor than the unphosphorylated enzyme.
129
Name the four kinases that phosphorylate and activate Tyrosine Hydroxylase (TH). What is the functional advantage of this?
Kinases: PKA, CaMKII, MAPK, and PKC.
Advantage: This is convergent signalling. Multiple pathways (e.g., from synaptic input, action potential firing, receptor tyrosine kinase activation) all converge on TH to ensure increased catecholamine synthesis.
130
Contrast the two mechanisms for regulating Tyrosine Hydroxylase (TH) activity in terms of speed and duration
Posttranslational Modification (Phosphorylation):
-Mechanism: Kinases (PKA, CaMK, etc.) directly phosphorylate existing TH protein.
-Effect: Fast but short-lived increase in activity.
Transcriptional Regulation (Gene Expression):
-Mechanism: CREB activation induces the transcription of the TH gene.
-Effect: Slower but more long-lived/sustained increase in activity due to more enzyme being present.
130
How is the level of the Tyrosine Hydroxylase (TH) enzyme itself regulated for a sustained increase in catecholamine synthesis?
The transcription of the TH gene is induced by the transcriptional activator CREB.
This leads to the synthesis of more TH enzyme, providing a long-term supply increase.
131
What is the relationship between the kinases that phosphorylate TH and the activator that induces its transcription?
The same kinases that phosphorylate TH (PKA, CaMK, MAPK) also activate CREB.
Therefore, a single signaling event (e.g., increased calcium) can both immediately activate existing TH enzyme and initiate the slower production of more TH enzyme.
132
Why can't the baseline activity of Tyrosine Hydroxylase (TH) support periods of high neuronal activity?
Under normal conditions, TH is somewhat active and generates a modest, baseline level of catecholamines (dopamine, norepinephrine, or epinephrine).
During strong activation, this modest rate of synthesis is insufficient to meet the increased demand caused by heightened neurotransmitter secretion.
133
What is NGF, what is its primary receptor, and what type of protein is this receptor?
NGF: Neurotrophic Growth Factor.
Receptor: TrkA.
Receptor Type: A Receptor Tyrosine Kinase (RTK).
134
What are the three key functions of NGF/TrkA signaling for sensory and sympathetic neurons during development?
Survival (prevents programmed cell death/apoptosis).
Differentiation (maturation into a specific neuronal type).
Neurite Outgrowth (growth of axons and dendrites).
135
What is the overarching principle demonstrated by the activation of TrkA by NGF?
Divergent Signalling. A single signal (NGF binding) activates multiple distinct signaling pathways (PLC, Ras/MAPK, PI3K/Akt) to initiate different cellular functions.
136
How does NGF/TrkA activation lead to MAPK pathway activation, and what is its primary function in neurons?
Mechanism: The adaptor protein Grb2 recruits a GEF, which activates Ras, leading to the MAPK pathway.
Function: Primarily involved in neuronal differentiation and neurite outgrowth.
137
How does NGF/TrkA activation lead to PLC pathway activation, and what is the result?
Mechanism: A different adaptor protein recruits and activates PLC (a specific isoform for RTKs).
Result: Generation of the second messengers IP₃ and DAG, leading to calcium release and PKC activation.
Function: Supports differentiation and neurite outgrowth
138
How does NGF/TrkA activation lead to PI3K/Akt pathway activation, and what is its critical, unique function?
Mechanism: A third adaptor protein initiates the PI3K/Akt kinase pathway. Akt is a serine/threonine kinase can phosphorylate a number of target proteins in the cytoplasm and the nucleus, where it translocated following activation.
Function: Cell survival; it prevents developing neurons from undergoing programmed cell death (apoptosis).
